Field of modular multifunctional ligands

ABSTRACT

This invention pertains to a surface ligand; preparation of the ligand; colloidal nanoparticle, such as quantum dot bearing one or more of the ligand; and a bioconjugate characterized by a nanoparticle bearing one or more of the ligand conjugated to a biomolecule. The ligand is characterized by the presence of a first module containing atoms that can attach to an inorganic surface; a second module that imparts water-solubility to the ligand and to the inorganic surface that may be attached to the ligand; and a third module that contains a functional group that can, directly or indirectly, conjugate to a biomolecule. Order of the modules can be different and other modules and groups can be on the ligand. Preparation of the ligand includes the steps of reacting a compound having atoms that can attach to an inorganic surface with a water-solubilizing compound that imparts the property of water-solubility to the ligand and the inorganic surface to which it may be attached and purification thereof. Colloidal nanoparticle is characterized by an inorganic surface having attached to it one or more of the ligands, The colloidal bioconjugate is characterized by an inorganic surface having attached thereto one or more of the ligand wherein at least some of the ligand have a biomolecule conjugated thereto.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of multifunctional ligands,colloidal nanoparticles and colloidal bioconjugates.

2. Description of Related Prior Art

Known methods to prepare water-soluble nanoparticles, such asdemiconductor nanoparticles (quantum dots) and matallic nanoparticles,involve the capping of those nanoparticles with ionized dihydrolipoicacid molecules. Electrostatic self-assembly techniques allow one toeasily prepare bioconjugates that take advantage of positively chargeddomains of proteins coupled to the negatively charged carboxylate groupson the nanoparticle surface. Some limitations to this approach includethe restriction of operating in basic environments and the inability toform direct covalently linked nanocrystal-biomolecule conjugates.

Other known approaches have used small organic surface ligands, such asmercaptoacetic acid and aminoethane thiol, to generate water-solublenanoparticles and other systems. The major disadvantage of such systemsinvolves the poor temporal stability of the nanoparticle ligands due tothe nature of the singly bound water-solubilizing groups which resultsin aggregated solutions after a short time. Water solubilization ofnanoparticles with hydrophilic dendritic structures and layer-by-layerassembly techniques has also been demonstrated with some degree ofsuccess. Most of these strategies provide for any pH stability, letalone long-term water solubility. acid and a polyethylene glycol,colloidal quantum dots and quantum ddot bioconjugates.

Advantages of the herein described and claimed invention include thefollowing: broad acidic and basic pH stability of the colloidalnanoparticles and colloidal bioconjugates: non-toxicity in and/or toliving cells of the colloidal nanoparticles and colloidal bioconjugates;non-aggregation in living cells of the colloidal nanoparticles andcolloidal bioconjugates; covalent conjugation of the ligand tobiomolecules; and control of biomolecular conjugation quantity,particularly when using mixed ligands.

Referring to FIG. 1, the ligand includes module #1, which includes agroup for attaching to an inorganic surface; module #2, which includes awater-solubilizing group that imparts water solubility and pH stabilityto the colloidal nanoparticle and colloidal bioconjugate; and module #3, which includes a functional group that allows conjugation to abiomolecule, directly or indirectly. It should be understood that theligand can have more than three modules and module order can bedifferent in that module #3 can be positioned before module #2 and itneed not be positioned at end of the ligand.

The surface interactive group or the attaching group of module #1 can beany chemical moiety having elements or chemical groups contained thereinthat are capable of binding to the surface of the inorganic particle.For example, the surface group includes, but is not limited to one ormore SH, NH₂, P, O or O═P. The attaching group of module #1 can containpassive components, such as alkylene groups, that do not apparentlycontribute to the function of the attaching group. A typical example ofa suitable compound that contains an alkylene group is an open thiocticacid which is composed of a five member ring containing two thiol atoms,a passive —CH₂ CH₂— alkylene group attached to a carboxyl-COOH endgroup. More than two thiol groups can be present in the acid, such as inthe ligand whose structural formula is shown in FIG. 2. In preparationof the ligand of FIG. 3, resorcinol is coupled to a mono tosylatedoligo(ethylene glycol) in a Mitsunobu ether synthesis. The resulting1,2-bis-oligo(ethylene glycol)benzene is modified in the Gabrielsynthesis to give the diamino species upon reaction with formalin andhydrochloric acid. Amide formation of the amines and the carboxylic acidof dihydrolipoic acid then results in the desired multi-dentate cappingligand shown in FIG. 3, where the repeating group “n” is typically inthe range of 3-100, more typically 4-10.

The functional end group of module #3 can be provided by the reaction ofan acid with a poly(ethylene glycol) or a polysacchride, such asdextrin. The functional group can be modified by known techniques.Ligands have been prepared with functional groups and groups thatinclude carboxylic, thiol, aryl and hydroxy groups that can be used inthe conjugation to proteins, DNA, RNA and other biologically relevantgroups.

The water solubilizing modular group of the designed ligand is basedupon well-established amphiphilic biocompatible poly(ethylene glycol)units. Because there is a wide variety of inexpensive, commerciallyavailable poly(ethylene glycols), enormous tailorability exists tomodify the solubility of the composite system. Furthermore, the modularnature of the poly(ethylene glycol) groups can be tailored to includecross-linkable subunits that can polymerize in the nanoparticle-ligandcomplex to form a core-protected encapsulated species that effectivelyshields it from the environment, a highly desirable trait for colloidalquantum dots, for example, in order to completely passivate the surfaceand ensure a high photoluminescence efficiency.

Typical preparation of a ligand is illustrated in FIG. 4 and involvesthe reaction of an acid and a poly(ethylene glycol). In a typicalsynthesis, commercially available thioctic acid and a 600 averagemolecular weight poly(ethylene glycol) are coupled in the presence ofdicyclohexylcarbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP) indichloromethane (DM) for several hours. Purification of the ligand canbe achieved by column chromatography on silica gel utilizing a mobilephase consisting of chloroform, methanol and acetone. The dithiane ringsystem of the material was reduced to the ring-opened dithiol in thepresence of sodium borohydride (NaBH₄) at room temperature. Purificationof the ligand was achieved through the use of silica gel chromatographyand similar mobile phase. Characterization of the ligand poly(ethyleneglycol) and dihydrolipoic acid, i.e., opened thioctic acid, was achievedthrough ¹H-NMR and ¹³C-NMR spectroscopy. Identification of the proposedspecies was determined by comparison to the parent subunits. Integrationratios of the poly(ethylene glycol) resonances to the dihydrolipoic acidbased resonances also confirmed the approximate chain length of thepoly(ethylene glycol).

It should be noted that FIG. 4 depicts a reaction wherein thepoly(ethylene glycol) is attached to the acid first and then the ringwith the two thiol atoms is opened, thus separating the thiol groups. Itis also possible to first open the ring and then attach thepoly(ethylene glycol). However, attachment and subsequent ring opening,shown in FIG. 4 is preferred since yield of the ligand is typicallyincreased from about 30% to about 80% and purification thereof is muchcleaner. However, a group can be provided in a known way at end of theligand of FIG. 4 which can directly conjugate to a biomolecule.

A colloidal nanoparticle is an inorganic nanoparticle attached to atleast one ligand. In general, the inorganic particle can be anyinorganic material exhibiting a distinct physical property that can beused to identify that material. The physical properties can be, but arenot limited to, emission such as photoluminescence, absorption,scattering and plasmon resonances. For example, the inorganic particlecan be illuminated with a light source at an absorption wavelength tocause an emission at an emission wavelength that can be used todistinguich the emitting material from other materials.

Examples of inorganic particles include, but are not limited to,inorganic colloids and semiconducting nanoparticles. The particles canbe metallic or magnetic. The particles can also be crystalline. Examplesof inorganic colloids include Ag, Au or a phosphor. The phosphor can bean inorganic phosphor, such as a rare earth oxide. The inorganiccolloids can exhibit distinct reflectivity and scattering properties,depending on the size of the particles in the colloid. Examples ofsemiconducting nanoparticles include compounds of groups II-VI, III-Vand IV of the Periodic Table. Elements from these groups include binaryCdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb, GaN, HgS, HgSe,HgTe, InAs, InP, InSb, InN, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge, Si, oran alloy or a mixture thereof, including ternary and quaternarymixtures. The semiconducting nanoparticles can be semiconductingnanocrystals or quantum dots. The nanocrystals can be illuminated with alight source at an absorption wavelength to cause an emission at adifferent wavelength. The emission has a frequency that corresponds to aband gap of quantum confined semiconductor material. The band gap is afunction of the size of the nanocrystal. Nanocrystals having smalldiameters can have properties intermediate between molecular and bulkforms of matter. For example, nanocrystals based on semiconductormaterials having small diameters can exhibit quantum confinement of boththe electron and hole in all three dimensions, which leads to anincrease in the effective band gap of the material with decreasingcrystallite size. Consequently, both the optical absorption and emissionof nanocrystals shift to the blue, i.e., to higher energies, as the sizeof the crystallites decreases. Of particular interest herein is the useof the nanoparticles, such as quantum dots, having thereon one or moreligands. Quantum dots having one or more ligands attached thereto arereferred to herein as colloidal quantum dots, indicating the ability todisperse them in solution, such as organic and aqueous environments.

The outer surface of the nanocrystal can include a layer of compoundsderived from the coordinating solvent used during the growth process.The surface can be modified by repeated exposure to an excess of acompeting coordinating group to remove the native ligand and replace itwith the new one. For example, a dispersion of the capped nanocrystalcan be treated with a coordinating organic compound, such as pyridine,to produce crystallites which disperse readily in pyridine, methanol,and aromatics but no longer disperse in aliphatic solvents. Such asurface exchange process can be carried out with any compound capable ofcoordinating to or bonding with the outer surface of the nanocrystal,including, for example, phosphines, thiols, amines and phosphates. Thenanocrystal can be exposed to short chain polymers which exhibit anaffinity for the surface and which terminate in a moiety having anaffinity for a suspension or dispersion medium. Such affinity improvesthe stability of the suspension and discourages flocculation of thenanocrystal.

Pursuant to prior art practice, nanoparticle size distribution duringthe growth stage can be estimated by monitoring the absorption linewidths of the particles. Modification of the reaction temperature inresponse to changes in the absorption spectrum or emission spectrum ofthe particles allows the maintenance of a sharp particle sizedistribution during growth. Reactants can be added to the nucleationsolution during crystal growth to grow larger crystals. By stoppinggrowth at a particular nanocrystal average diameter and choosing theproper composition of the semiconducting material, the emission spectraof the nanocrystals can be tuned continuously over the wavelength rangeof 400 to 800 nm. The nanocrystal has a diameter of less than 150 Å. Apopulation of nanocrystals has average diameters in the range of 15 Å to125 Å. In a quantum dot, the core is typically in the range of 15-125 Å,more typically 20-40 Å, and the coating thickness is typically in therange of 2-100 Å, more typically 5-30 Å.

Transmission electron microscopy or small angle x-ray scattering canprovide information about the size, shape and distribution of thenanocrystal population. Powder x-ray diffraction patterns can providethe most complete information regarding the type and quality of thecrystal structure of the nanocrystals. Estimates of size are alsopossible since particle diameter is inversely related, via the x-raycoherence length, to the peak width. For example, the diameter of thenanocrystal can be measured directly by transmission electron microscopyor estimated from x-ray diffraction data using, for example, the Schererequation. It also can be estimated from the UV/Vis absorption spectrum.

Preparation of the quantum dots bearing water-solubledihydrolipoate-poly(ethylene glycol) ligands can be accomplished inseveral steps. Typically, a small volume of the quantum dot solution inthe native trioctylphosphine/trioctylphosphine oxide is precipitatedwith methanol and centrifuged to remove the excess trioctyphosphine- andphosphineoxide. An excess of the poly(ethylene glycol) modifieddihydrolipoic acid is added (either neat of in methanol or ethanol) tothe precipitated quantum dots and the system is evacuated and backfilledwith an atmosphere of nitrogen. The quantum dots readily disperse in theneat ligand (or a solution containing the ligand) with gentle heatingand the solution is allowed to stir for several hours. The solution isthen further diluted with a small volume of ethanol and precipitatedwith hexanes and chloroform to ensure a mono-phasic solution in whichthe quantum dots are precipitated. The precipitate is concentrated bycentrifuging and the supernatant is discarded. The process is repeated 2to 3 times and the sample is then dried under a gentle stream ofnitrogen. The quantum dots are dispersed in water and purified through acentrifugal filtration device with a nominal molecular weight cutoff at50,000 and filtered through a 0.45 μm PTFE frit. The water solublequantum dots are then transferred to aqueous buffer solutions at variouspHs and remain aggregate-free for extended periods of time of manymonths.

FIG. 5 is a depiction of a colloidal quantum dot CdSe/ZnS wherein m is6, indicating 6 separating poly(ethylene oxide) repeating units. Itshould be understood that the colloidal quantum dot of FIG. 5 can bearmany, i.e., hundreds and thousands, of the surface ligands. Also, m canbe larger than 6.

FIG. 6 shows a mixed surface ligand composition of dihydrolipoic acid todihydrolipoic acid-poly(ethylene glycol) 600 (PEG) on the quantum dot.Aqueous solutions with ratios using 1:0 to 0:1 of dihydrolipoic acid(DHLA) to DHLA-PEG (600) surface ligands were prepared.

FIG. 7 schematically depicts a reaction of a mixed ligand colloidalquantum dot with a DNA biomolecule in presence of ethylene1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) andN-hydroxysulfosuccinimide (sulfo-NHS). Duration of the biomolecule DNAconjugation is typically 10-30 minutes and it can be carried out at roomtemperature.

FIG. 8 shows a luminescence image of sets of two solutions of quantumdots capped with the dihydrolipoic acid-polyethylene glycol (600) ofthis invention at various pHs compared to solution of quantum dotscapped with a prior art compound.

The pH variation is based on experiments of incubating the quantum dotscoated with poly(ethylene glycol)-modified dihydrolipoic acid ligands atpHs of 5 to 8, which suggests that luminescence of such systems arequite stable and unaltered from prior art. Luminescence experiments wereconducted with CdSe/ZnS quantum dots coated with DHLA-PEG (600) inbuffered aqueous solutions. The samples were excited with a UV lamp at365 nm and the emission was centered at 553 nm. The use of mixed ligandstrategy is not limited to dihydrolipoic acid, since others can be used.

In practice, the mixed surface ligand is typically used since the group(—COOH) can be directly conjugated to a biomolecule. The mixed ligandapproach is also typically used since it allows for better control ofthe quantity of the biomolecule that can be provided on an inorganicsurface of nanoparticles or quantum dots. Advantages of using mixedsurface ligands include control of functional groups on thenanoparticle; makes solubility and coupling to biomolecules two separateissues; allows one to use a multiple of functional groups withoutaffecting solubility; and allows one to titrate down the number ofcoupled molecules to 3, 2, or even 1.

1. A ligand comprising a first module that contains a group that canattach to an inorganic surface; a second module that can impartwater-solubility to said ligand and to said inorganic surface to whichsaid ligand may be attached; and a third module that contains afunctional group.
 2. The ligand of claim 1 wherein said attaching groupof said first module consists of atoms and groups of atoms selected fromthe group consisting of SH, NH₂, P, O, O═P, and mixtures thereof.
 3. Theligand of claim 2 wherein said attaching group of said first module isat least two thiol atoms.
 4. The ligand of claim 1 wherein saidattaching group of said first module is two adjacent thiol groups in afive member ring.
 5. The ligand of claim 2 wherein said functional groupis selected from the group consisting of hydroxyl, carboxyl, amino,aryl, and sulfhydryl groups.
 6. The ligand of claim 5 wherein saidsecond module includes water-solubilizing repeating units of compoundsselected from the group consisting of poly(ethylene glycol),polysaccharides, and mixtures thereof.
 7. The ligand of claim 6 whereinnumber of said repeating units from derived poly(ethylene glycol) isfrom about 3 to about
 100. 8. The ligand of claim 1 wherein saidattaching group of said first module is two adjacent thiol atoms in anopen five member ring attached to an alkylene moiety of 2 to 4 carbonatoms which in turn is attached to the ester group (—CO—O—); said secondmodule consists of one or more ethylene oxide repeating units derivedfrom poly(ethylene glycol); and said functional group in said thirdmodule is a hydroxyl group (—OH).
 9. The ligand of claim 6 wherein saidattaching group of said first module is two adjacent thiol atoms in anopen five member ring attached to an alkylene moiety of 2 carbon atomswhich in turn is attached to the ester group (—CO—O—); said secondmodule consists of 4 to 10 ethylene oxide repeating units derived frompoly(ethylene glycol); and said functional group of said third module iscarboxyl group (—COOH).
 10. The ligand of claim 9 wherein said attachinggroup of said first module is two pairs of adjacent thiol groups witheach pair of the thiol group being members of an open five member ring,the opening in each pair being between the thiol group.
 11. A method forpreparing a ligand comprising the step of reacting an acid having agroup that can attach to an inorganic surface with a water-solubilityimparting group, the ligand comprising a first module that contains agroup that can attach to an inorganic surface; a second module that canimpart water-solubility to said ligand and to said inorganic surface towhich said ligand may be attached; and a third module that contains afunctional group.
 12. The method of claim 11 including the step ofpurifying the ligand.
 13. The method of claim 12 wherein the attachinggroup consists of atoms and groups of atoms selected from the groupconsisting of SH, NH₂, P, O, O═P, and mixtures thereof; thewater-solubility imparting moiety is derived from compounds selectedfrom the group consisting of poly(ethylene glycol), polysaccharide, andmixtures thereof; and the functional group is selected from the groupconsisting of hydroxyl, carboxyl, amino, aryl, and sulfsulfhydrylgroups.
 14. The method of claim 11 wherein the attaching group consistsof atoms and groups of atoms selected from the group consisting of S, N,P, O, O═P, and mixtures thereof; the water-solubility imparting moietyis selected from ethylene oxide repeating units derived frompoly(ethylene glycol); and the functional group is selected fromfunctional groups capable of direct covalent conjugation to abiomolecule.
 15. The method of claim 12 wherein the attaching group ofthe acid is a pair of thiol atoms in a five-member ring attached to acarboxyl group and the water-solubility imparting moiety is more thanone repeating units derived from poly(ethylene glycol).
 16. The methodof claim 15 wherein said step of reacting is conducted in presence ofdicyclohexylcarbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP) indichloromethane (DM) to form a pre-ligand wherein the attaching group isin a closed five-member ring.
 17. The method of claim 16 including thestep of opening the five-member ring by continuing said reacting step inthe presence of sodium borohydride (NaBH₄).
 18. The method of claim 17wherein said reacting step is carried out at room temperature.
 19. Acolloidal semiconducting nanoparticle comprising an inorganicnanoparticle having at least one ligand attached thereto, said ligandcomprising a first module that contains a group that can attach to aninorganic surface; a second module that can impart water-solubility tosaid ligand and to said inorganic surface to which said ligand may beattached; and a third module that contains a functional group.
 20. Thecolloidal nanoparticle of claim 19 wherein said nanoparticle is acompound and/or an element selected from the group consisting of II-VI,III-V, and VI compounds and/or elements of the periodic Table; whereinthe attaching group consists of atoms and groups of atoms selected fromthe group consisting of SH, NH₂, P, O, O═P, and mixtures thereof. 21.The colloidal nanoparticle of claim 20 wherein said compounds and/orelements are selected from the group of binary semiconductors consistingof CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb, GaN, HgS,HgSe, HgTe, InAs, InP, InSb, InN, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge,Si, ternary semiconductors and mixtures thereof; wherein said attachinggroup of the first module is at least two thiol atoms.
 22. The colloidalnanoparticle of claim 21 wherein said nanoparticle is a quantum dotcomposed of a core and a coating thereon; wherein said attaching groupof the first module is two adjacent thiol atoms in a five member ring.23. The colloidal nanoparticle of claim 22 wherein said functional groupis selected from the group consisting of hydroxyl, carboxyl, amino,aryl, and sulfhydryl groups.
 24. The colloidal nanoparticle of claims 23wherein the second module includes water-solubilizing repeating units ofcompounds selected from the group consisting of poly(ethylene glycol),polysaccharide, and mixtures thereof.
 25. The colloidal nanoparticle ofclaim 24 wherein number of said repeating units derived frompoly(ethylene glycol) is from about 3 to about
 100. 26. The colloidalnanoparticle of claim 25 wherein said attaching group of said firstmodule is two adjacent thiol atoms in an open five member ring attachedto an alkylene moiety of 2 to 4 carbon atoms which in turn is attachedto said ester group (—CO—O—); said second module consists of one or moreethylene oxide repeating units derived from poly(ethylene glycol); andsaid functional group in said third module is a hydroxyl group (—OH).27. The colloidal nanoparticle of claim 26 wherein said attaching groupof said first module is two adjacent thiol atoms in an open five memberring attached to an alkylene moiety of 2 carbon atoms which in turn isattached to said ester group (—CO—O—); said second module consists of 4to 10 ethylene oxide repeating units; and said functional group of saidthird module is carboxyl group (—COOH)
 28. The colloidal nanoparticle ofclaim 27 wherein the attaching group of the first module is two pairs ofadjacent thiol atoms with each pair of the thiol atoms being members ofan open five-member ring, the opening in each pair being between thethiol atoms.
 29. The colloidal particle of claim 28 wherein the core ofsaid quantum dot is CdSe and the coating thereon is ZnS.
 30. Abioconjugate comprising an inorganic nanoparticle having attachedthereto at least one ligand comprising a first module that contains agroup that can attach to an inorganic surface; a second module that canimpart water-solubility to said ligand and to said inorganic surface towhich said ligand may be attached; and a third module that contains afunctional group; and a biomolecule attached to said functional group.31. The bioconjugate of claim 30 wherein said nanoparticle is a compoundand/or an element selected from the group consisting of II-VI, III-V,and VI compounds and/or elements of the periodic Table; wherein theattaching group consists of atoms and groups of atoms selected from thegroup consisting of S, N, P, O, O═P, and mixtures thereof.
 32. Thebioconjugate of claim 31 wherein said nanoparticle is composed ofcompounds selected from the group consisting of CdS, CdSe, CdTe, ZnS,ZnSe, ZnTe, MgTe, GaAs, GaP, GaSb, GaN, HgS, HgSe, HgTe, InAs, InP,InSb, InN, AlAs, AlP, AlSb, AlS, PbS, PbSe, Ge, Si, and mixturesthereof; wherein said attaching group of the first module is at leasttwo thiol atoms.
 33. The bioconjugate of claim 32 wherein saidbiomolecule is DNA.
 34. The bioconjugate of claim 32 wherein saidattaching group of said first module is two adjacent thiol atoms in anopen five-member ring attached to an alkylene moiety of 2 to 4 carbonatoms; said second module consists of one or more ethylene oxiderepeating units derived from poly(ethyle glycol); and said functionalgroup in said third module is a hydroxyl group (—OH).
 35. Thebioconjugate of claim 34 wherein said alkylene moiety is 2 carbon atoms;said second module consists of 4 to 10 ethylene oxide repeating units;and said functional group of said third module is carboxyl group(—COOH).